research article antimelanoma and antityrosinase from alpinia galangal...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 186505 5 pageshttpdxdoiorg1011552013186505

Research ArticleAntimelanoma and Antityrosinase fromAlpinia galangal Constituents

Chih-Yu Lo1 Po-Len Liu2 Li-Ching Lin3 Yen-Ting Chen3 You-Cheng Hseu4

Zhi-Hong Wen5 and Hui-Min Wang36

1 Department of Food Science National Chiayi University Chiayi 60083 Taiwan2Department of Respiratory Therapy College of Medicine Kaohsiung Medical University Kaohsiung 80708 Taiwan3Department of Fragrance and Cosmetic Science Kaohsiung Medical University 100 Shih-Chuan 1st RoadSan-Ming District Kaohsiung 80708 Taiwan

4Department of Cosmeceutics College of Pharmacy China Medical University Taichung 40402 Taiwan5Department of Marine Biotechnology and Resources National Sun Yat-sen University Kaohsiung 80424 Taiwan6Graduate Institute of Natural Products Kaohsiung Medical University Kaohsiung 80708 Taiwan

Correspondence should be addressed to Hui-Min Wang davidwkmuedutw

Received 22 April 2013 Accepted 28 May 2013

Academic Editors H-W Chang L-Y Chuang S Guleria and S Yasmin

Copyright copy 2013 Chih-Yu Lo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Two compounds 17-bis(4-hydroxyphenyl)-146-heptatrien-3-one (BHPHTO) and bisdemethoxycurcumin (BDMC) they havebeen isolated from the rhizomes of Alpinia galangal and the structures of both pure constituents were determined usingspectroscopic analyses The study examined the bioeffectivenesses of the two compounds on the human melanoma A2058 andshowed that significantly inhibited the proliferation of melanoma cells in the cell viability assay This research was also taken onthe tests to B16-F10 cell line and showed minor inhibitory consequences of cellular tyrosinase activities and melanin contents Ourresults revealed the anticancer effects of A galangal compounds and therefore the target compounds could be potentially appliedin the therapeutic application and the food industry

1 Introduction

Alpinia galangal belongs to the Zingiberaceae family and theherb grows mainly in South East Asia It is now cultivatedthroughout tropical and subtropical Asia such as IndiaEgypt Thailand Malaysia Indonesia and China The herbsare usually not only used for seasoning but also for traditionalmedicine The rhizomes contain essential oil Many essentialoils were obtained by hydrodistillation The yield of essentialoils ranged from 132 to 0143 [1ndash3]Thewide range ofmajorvolatile compounds was identified and characterized by GCand GCMS as endo-fenchyl acetate zerumbone 18-cineoleand myrcene For A galangal rhizome chemical studiesa group of related phenylpropanoids was identified [4 5]Among these phenylpropanoids 11015840S-11015840-acetoxychavicol ace-tate (galangal acetate) was the most studied and reported topossess various activities such as antioxidative antifungal

antitumor and anti-inflammatory activities However onlysome studies for A galangal grown in Taiwan were published[6] Human skin is normally contacted with damage stresswhich is produced by external and intrinsic sources such asultraviolet (UV) radiation free radicals and reactive oxygenspecies [7] There are many studies about the skin exposedto oxidative stress or UV radiation and are responsible foraging or tumorigenesis [8] Melanoma a malignant tumorof epidermal melanocytes is one of the most deadly skincancers Within the past several decades the occurrences ofcutaneousmalignantmelanomahave increased because it hasa strong propensity tometastasize and therefore is one of themost aggressive skin cancers Unlike other cancersmalignantmelanoma is not easy to treat with surgery radiotherapyor chemotherapy A good chemotherapeutic agent will bea naturally occurring agent and can induce cytotoxicity incancer cells

2 The Scientific World Journal

In mammals skin hair and eyes darkening is deter-mined by the synthesis and distribution ofmelanin In skin itis amixture of pigmented biopolymers that is synthesized in aunique organelle the melanosome of melanocytes Excessivebiosynthesis of melanin induces various related pigmentdisorders such as senile lentigo melasma freckles and pig-mented acne scars that are of particular concern to women aswell as menTheir treatment usually involves the use of med-icines or medicinal cosmetics containing depigmenting orskin-whitening components Safe and effective regulators thatact tominimize skin pigmentation abnormalities include nat-ural and synthetic depigmenting agents However only a feware used as therapeutic agents primarily because of varioussafety concerns and lowwhitening bioactivity Inmelanogen-esis L-tyrosine is hydroxylated to dihydroxyphenylalanine(L-DOPA) and then L-DOPA is oxidized to DOPA-quinonewith two initial steps [9] Pigment coloring hair skin andeyes because of the key protein tyrosinase is recognized tobe the first two and rate-limiting enzyme in the biosynthesisof melanins [10] Recently much attention has been drawn tothe application of tyrosinase inhibitors to medical treatmentsand cosmetic businesses Therefore in clinical usage tyrosi-nase inhibitors are being taken for dermatological disordertreatments related to melanin hyperaccumulation and arethus fundamental in cosmetics for depigmentation [11]

2 Materials and Methods

21 Reagents and Materials All solvents were at analyticalgrade Lipophilic Sephadex (LH-20) resin was purchasedfrom Sigma-Aldrich Inc (St Louis MO USA) Reversephase (C18 25ndash40120583m) sorbent was purchased from MerckChemical Co (Darmstadt Germany)

22 Extraction and Purification of BHPHTO and BDMC Therhizomes of A galangal were collected from the Native PlantEcological Garden in National Chiayi University The air-dried rhizomes (300 g) of A galangal were ground into a finepowder and extracted successively with 95 ethanol atroom temperature The crude ethanol extract (CEE) wasfiltered and evaporated to slurry using a rotary evaporatorThe slurry was then suspended with water and partitionedsuccessively three times with hexane (times3) and ethyl acetate(times3) The hexane and ethyl acetate extracts were separatelydried using a rotary evaporator at 40∘C followed by freeze-drying for 48 h to give dried hexane and ethyl acetate extract(AG-EtOAC) respectively The AG-EtOAC fraction was firstsubjected to passage over LH-20 using 95 ethanol Theeluted solution was collected by test tubes Only the tubesthat contained major components were concentrated andfollowed by eluting isocratically in the self-pack C18 with80 methanol solution to obtain BHPHTO and BDMCTheidentities of 17-bis(4-hydroxyphenyl)-146-heptatrien-3-one(BHPHTO) and bisdemethoxycurcumin (BDMC) (Figure 1)were confirmed by comparison of theirNMRandMS spectraldata with those available in the literature [12]

O

HO OH

17-Bis(4-hydroxyphenyl)-146-heptatrien-3-one (BHPHTO)

(a)

O

HO OH

O

Bisdemethoxycurcumin (BDMC)

(b)

Figure 1 The structure of 17-bis(4-hydroxyphenyl)-146-hepta-trien-3-one (BHPHTO) and bisdemethoxycurcumin (BDMC)

23 Human Melanoma A2058 Cell and Mouse B16-F10 Mel-anoma Cell Cultures The human skin cancer A2058 celllines were derived and purchased from the Bioresource Col-lection and Research Center (BCRC number 60240 Hsin-chu Taiwan ROC) the American Type Culture Collection(ATCCnumber CRL-11147Manassas VAUSA)ThehumanmetastaticmelanomaA2058 cell line is derived from 43-year-old male [13]

And the melanoma B16-F10 cells (BCRC 60031) wereobtained from ATCC (Manassas VA USA) cultured inDMEM (134mgmL Dulbeccorsquos modified Eaglersquos medium10mM HEPES 143UmL benzylpenicillin potassium100mgmL streptomycin sulfate and 24mM NaHCO

3 pH

71) containing 10 FBS 1 PS and incubated at 37∘C under5 CO

2atmosphere [14]

24 Cell Proliferation Assay Theeffects of compounds on cellgrowths were according to the MTT assay procedures [15]The method is based on the ability of a mitochondrial dehy-drogenase from viable cells to cleave the tetrazolium ringsof the pale yellow MTT and form impermeable crystals ofa dark-blue formazan thus resulting in accumulation withinhealthy cells Briefly cells were seeded in 96-well plates at adensity of 8 times 103 cellswell The medium was then changedand cells were maintained in either solvent alone (controlcells) or in the presence of the indicated each sample in a finalvolume of 100 120583L in 10 FBS culture medium Each samplewas added to a microplate and incubated under the sameconditions as above for 24 h After 24 h of incubation themediumwas replaced with 100 120583L of freshmedium including05mgmL MTT The plate was cultured in a 37∘C incubatorfilled with 5 CO

2for 2 h Each precipitate in a specific

dish was dissolved in 100 120583L of DMSO to dissolve the purpleformazan crystals After the dishes were gently shaken for10min in the dark to ensuremaximal dissolution of formazancrystals the absorbance (119860) values of the supernatant were

The Scientific World Journal 3

0

30

60

90

120

Control 1 5 10 25 50 100

A20

58 ce

ll vi

abili

ty (

of c

ontr

ol )

BHPHTO (120583M)

lowastlowast

lowast

(a)

0

30

60

90

120

Control 1 5 10 25 50 100

A20

58 ce

ll vi

abili

ty (

of c

ontr

ol)

BDMC (120583M)

lowast

lowast

lowast

(b)

Figure 2 The effects of ginger extracts (BHPHTO and BDMC) onA2058 cell growths measured by MTT assay The data representedthemean valueplusmn SDof triplicate values for three independent exper-iments Comparisons were subjected to Studentrsquos 119905-test Significantlydifferent at lowast119875 lt 005

measured at 595 nm (UV vis BioTek Winooski VT) Cellgrowth was calculated as

Cell viability () =(119860 sample minus 119860 blank )

(119860control minus 119860blank)times 100 (1)

25 Measurement of B16-F10 Cellular Tyrosinase ActivityTesting cell tyrosinase activity in vitro B16-F10 melanomacells (105 cells per well) were placed in 24-well plates in 300 120583Lof medium containing various concentrations of testing sam-ples and incubated for 2 days [16] The sample-treated cellswere washed with phosphate-buffered saline (PBS) and lysedwith 1 Triton X-100PBS The enzyme extract of cellularlysate was added to 50120583L of 2mM L-tyrosine This reactionwas then incubated at 37∘C for 3 h in a dark environment andthe absorbance at 490 nm was measured on a spectropho-tometer

26 Determination of B16-F10 Cellular Melanin ContentsBriefly we followed the previous method with minor mod-ifications [17] Cell pellets were dissolved in 20N NaOHcontaining 10 DMSO and heated at 90∘C for 1 h andsuspensions were clarified by centrifugation for 10min at

0

20

40

60

80

100

120

Control 1 5 10 25 50 100

Cel

l via

bilit

y (

of c

ontro

l)

BHPHTO (120583M)

lowast lowast

lowast

lowast

(a)

Control 1 5 10 25 50 100BDMC (120583M)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowast

lowast

lowast

(b)

Figure 3The impact of BHPHTOandBDMC toB16-F10melanomacell viabilities The data represented the mean value plusmn SD oftriplicate values for three independent experiments Comparisonswere subjected to Studentrsquos 119905-test Significantly different at lowast119875 lt 005

10000timesg The amount of melanin was determined spec-trophotometrically based on the absorbance at 475 nm [18]

27 Statistical Analysis The data were expressed as the meanvalue obtained in three experiments Statistical comparisonswere performed by Studentrsquos 119905-test for paired values

3 Results and Discussion

31 Cellular Proliferative Depressing Effects of BHPHTO andBDMC on Melanoma Cells The main reason of the death ofa patient is due to the tumor proliferation and metastasis[12] Therefore it is urgent to find valuable and significantnovel biomedicinal components for anticancer therapiesMTT assay illustrated the anticell proliferation of two kindsof human skin melanoma cells A2058 cells for 24 h treat-ments In Figure 2 to evaluate the effects of several ginger(BHPHTO and BDMC) extracts on cancer cytotoxicity cellswere treated with various concentrations from 0 to 100 120583MWe demonstrated the cytotoxic abilities on human skinmelanoma A2058 cells of the extracts and cellular prolifer-ations were inhibited in dose-dependent manner when cells


0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BHPHTO (120583M)

lowastlowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BDMC (120583M)

lowastlowast

(b)

Figure 4 The tyrosinase activity of B16-F10 cells treated with vari-ous concentrations of BHPHTO and BDMC The data representedthemean valueplusmn SDof triplicate values for three independent exper-iments Comparisons were subjected to Studentrsquos 119905-test Significantlydifferent at lowast119875 lt 005

were exposed to a high dose of all extracts BHPHTO andBDMC (50 120583M) and cell viabilities exhibited less than 25after 24 h of treatment

32 Cytotoxicity of BHPHTO and BDMC in B16-F10 Cells 3-(45-Dimetylthiazol-2-yl)-25-diphenyl tetrazolium bromide(MTT) assay was investigated to study the cytotoxicities ofBHPHTO and BDMC in B16F10 cells The samples weretreated with various concentrations from 1 120583M to 100 120583Mand the vehicle control group had no testing agents with 05DMSO As shown in Figure 3 With 25120583M BHPHTO about55B16-F10 cells remained andwith 25 120583MBDMC still 65B16-F10 cells remained

33 B16-F10 Cellular Tyrosinase Activity and Melanin Contentof BHPHTO and BDMC Melanin is a vitally important fac-tor in determining the skin color of human The melano-genesis pathway consists of the enzymatic L-tyrosine hydrox-ylation and the oxidation of L-dopa to its correspondingdopaquinone [9] After the two tyrosinase-catalyzed stepsadditional multiple biosynthesis steps followed and yielded

0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BHPHTO (120583M)

lowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BDMC (120583M)

lowast

lowastlowast

lowast

(b)

Figure 5 The melanin content of B16-F10 cells treated with variousconcentrations of BHPHTO and BDMC The data represented themean value plusmn SD of triplicate values for three independent experi-ments Comparisons were subjected to Studentrsquos 119905-test Significantlydifferent at lowast119875 lt 005

melanin [10] We tried to investigate if BHPHTO and BDMChave mouse B16F10 cellular tyrosinase-inhibiting abilities(Figure 4) and melanin content (Figure 5) decreasing power[11] Although the tyrosinase activity of B16-F10 resultsshowed that with 5 or 10 120583M of BHPHTO and BDMC thereis no obvious variation and with concentrations higher than25 120583M tyrosinase activity decreased for low cell viabilityBut interestingly even the cell viability is low the tyrosi-nase activity is completely high Melanin content howeverdid not show evident reduction and on the contrary withhighest concentrations of BHPHTO and BDMC at 25 120583Mthe melanin content decreased for low cell viability As thetendency of tyrosinase activity melanin content is completelyhigher than cell viability

Conflict of Interests

No contributing author has a conflict of interests in the publi-cation of this study


Authorsrsquo Contribution

Chih-Yu Lo and Po-Len Liu contributed equally to this work

Acknowledgment

The work was supported by the National Science Councilunder the Grant nos NSC 99-2313-B-415-006-MY3 and NSC99-2221-E-037-006-MY3

References

[1] K Saeio W Chaiyana and S Okonogi ldquoAntityrosinase andantioxidant activities of essential oils of edibleThai plantsrdquoDrugDiscoveries andTherapeutics vol 5 no 3 pp 144ndash149 2011

[2] M M Yusoff H Ibrahim and N A Hamid ldquoChemical charac-terization and antimicrobial activity of rhizome essential oils ofvery closely allied zingiberaceae species endemic to borneoAlpinia ligulata K SCHUM and Alpinia nieuwenhuizii VALrdquoChemistry and Biodiversity vol 8 no 5 pp 916ndash923 2011

[3] R C Padalia R S Verma V Sundaresan and C S ChanotiyaldquoChemical diversity in the genus Alpinia (Zingiberaceae) com-parative composition of courAlpinia species grown inNorthernIndiardquo Chemistry and Biodiversity vol 7 no 8 pp 2076ndash20872010

[4] HMatsuda Y Pongpiriyadacha TMorikawaM Ochi andMYoshikawa ldquoGastroprotective effects of phenylpropanoids fromthe rhizomes ofAlpinia galanga in rats structural requirementsand mode of actionrdquo European Journal of Pharmacology vol471 no 1 pp 59ndash67 2003

[5] TMorikawa S AndoHMatsuda S KataokaOMuraoka andM Yoshikawa ldquoInhibitors of nitric oxide production from therhizomes of Alpinia galanga structures of new 8-91015840 linked neo-lignans and sesquineolignanrdquo Chemical and PharmaceuticalBulletin vol 53 no 6 pp 625ndash630 2005

[6] T Noro T Sekiya M Katoh et al ldquoInhibitors of xanthine oxi-dase from Alpinia galangardquo Chemical and Pharmaceutical Bul-letin vol 36 no 1 pp 244ndash248 1988

[7] H-MWang C-Y Chen and Z-H Wen ldquoIdentifying melano-genesis inhibitors from Cinnamomum subaveniumwith in vitroand in vivo screening systems by targeting the human tyrosi-naserdquo Experimental Dermatology vol 20 no 3 pp 242ndash2482011

[8] S Briganti and M Picardo ldquoAntioxidant activity lipid perox-idation and skin diseases whatrsquos newrdquo Journal of the EuropeanAcademy ofDermatology andVenereology vol 17 no 6 pp 663ndash669 2003

[9] W T Liao T S Huang C C Chiu et al ldquoBiological propertiesof acidic cosmetic water from seawaterrdquo International Journal ofMolecular Sciences vol 13 no 5 pp 5952ndash5971 2012

[10] C-C Chiu H-L Chou P-F Wu H-L Chen H-M Wangand C-Y Chen ldquoBio-functional constituents from the stems ofLiriodendron tulipiferardquoMolecules vol 17 no 4 pp 4357ndash43722012

[11] L Kolbe T Mann W Gerwat et al ldquo4-n-butylresorcinol ahighly effective tyrosinase inhibitor for the topical treatmentof hyperpigmentationrdquo Journal of the European Academy ofDermatology and Venereology vol 27 supplement 1 pp 19ndash232013

[12] H Mohamad N H Lajis F Abas et al ldquoAntioxidative con-stituents of Etlingera elatiorrdquo Journal of Natural Products vol68 no 2 pp 285ndash288 2005

[13] H-M Wang C-C Chiu P-F Wu and C-Y Chen ldquoSub-amolide E from Cinnamomum subavenium induces sub-G1cell-cycle arrest and caspase-dependent apoptosis and reducesthe migration ability of human melanoma cellsrdquo Journal ofAgricultural and Food Chemistry vol 59 no 15 pp 8187ndash81922011

[14] H-M Wang C-Y Chen C-Y Chen et al ldquo(-)-N-Formylan-onaine fromMichelia alba as a human tyrosinase inhibitor andantioxidantrdquoBioorganic andMedicinal Chemistry vol 18 no 14pp 5241ndash5247 2010

[15] B-H Chen H-W Chang H-M Huang et al ldquo(-)-Anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[16] H-M Wang Y-T Chou Z-L Hong et al ldquoBioconstituentsfrom stems of Synsepalum dulcificum Daniell (Sapotaceae)inhibit human melanoma proliferation reduce mushroomtyrosinase activity and have antioxidant propertiesrdquo Journal ofthe Taiwan Institute of Chemical Engineers vol 42 no 2 pp204ndash211 2011

[17] Y-P Lin F-L Hsu C-S Chen J-W Chern and M-H LeeldquoConstituents from the Formosan apple reduce tyrosinaseactivity in human epidermal melanocytesrdquo Phytochemistry vol68 no 8 pp 1189ndash1199 2007

[18] J Y Jang J H Lee B W Kang K T Chung Y H Chol and BT Choi ldquoDichloromethane fraction of Cimicifuga heracleifoliadecreases the level of melanin synthesis by activating theERK or AKT signaling pathway in B16F10 cellsrdquo ExperimentalDermatology vol 18 no 3 pp 232ndash237 2009

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


In mammals skin hair and eyes darkening is deter-mined by the synthesis and distribution ofmelanin In skin itis amixture of pigmented biopolymers that is synthesized in aunique organelle the melanosome of melanocytes Excessivebiosynthesis of melanin induces various related pigmentdisorders such as senile lentigo melasma freckles and pig-mented acne scars that are of particular concern to women aswell as menTheir treatment usually involves the use of med-icines or medicinal cosmetics containing depigmenting orskin-whitening components Safe and effective regulators thatact tominimize skin pigmentation abnormalities include nat-ural and synthetic depigmenting agents However only a feware used as therapeutic agents primarily because of varioussafety concerns and lowwhitening bioactivity Inmelanogen-esis L-tyrosine is hydroxylated to dihydroxyphenylalanine(L-DOPA) and then L-DOPA is oxidized to DOPA-quinonewith two initial steps [9] Pigment coloring hair skin andeyes because of the key protein tyrosinase is recognized tobe the first two and rate-limiting enzyme in the biosynthesisof melanins [10] Recently much attention has been drawn tothe application of tyrosinase inhibitors to medical treatmentsand cosmetic businesses Therefore in clinical usage tyrosi-nase inhibitors are being taken for dermatological disordertreatments related to melanin hyperaccumulation and arethus fundamental in cosmetics for depigmentation [11]

2 Materials and Methods

21 Reagents and Materials All solvents were at analyticalgrade Lipophilic Sephadex (LH-20) resin was purchasedfrom Sigma-Aldrich Inc (St Louis MO USA) Reversephase (C18 25ndash40120583m) sorbent was purchased from MerckChemical Co (Darmstadt Germany)

22 Extraction and Purification of BHPHTO and BDMC Therhizomes of A galangal were collected from the Native PlantEcological Garden in National Chiayi University The air-dried rhizomes (300 g) of A galangal were ground into a finepowder and extracted successively with 95 ethanol atroom temperature The crude ethanol extract (CEE) wasfiltered and evaporated to slurry using a rotary evaporatorThe slurry was then suspended with water and partitionedsuccessively three times with hexane (times3) and ethyl acetate(times3) The hexane and ethyl acetate extracts were separatelydried using a rotary evaporator at 40∘C followed by freeze-drying for 48 h to give dried hexane and ethyl acetate extract(AG-EtOAC) respectively The AG-EtOAC fraction was firstsubjected to passage over LH-20 using 95 ethanol Theeluted solution was collected by test tubes Only the tubesthat contained major components were concentrated andfollowed by eluting isocratically in the self-pack C18 with80 methanol solution to obtain BHPHTO and BDMCTheidentities of 17-bis(4-hydroxyphenyl)-146-heptatrien-3-one(BHPHTO) and bisdemethoxycurcumin (BDMC) (Figure 1)were confirmed by comparison of theirNMRandMS spectraldata with those available in the literature [12]

O

HO OH

17-Bis(4-hydroxyphenyl)-146-heptatrien-3-one (BHPHTO)

(a)

O

HO OH

O

Bisdemethoxycurcumin (BDMC)

(b)

Figure 1 The structure of 17-bis(4-hydroxyphenyl)-146-hepta-trien-3-one (BHPHTO) and bisdemethoxycurcumin (BDMC)

23 Human Melanoma A2058 Cell and Mouse B16-F10 Mel-anoma Cell Cultures The human skin cancer A2058 celllines were derived and purchased from the Bioresource Col-lection and Research Center (BCRC number 60240 Hsin-chu Taiwan ROC) the American Type Culture Collection(ATCCnumber CRL-11147Manassas VAUSA)ThehumanmetastaticmelanomaA2058 cell line is derived from 43-year-old male [13]

And the melanoma B16-F10 cells (BCRC 60031) wereobtained from ATCC (Manassas VA USA) cultured inDMEM (134mgmL Dulbeccorsquos modified Eaglersquos medium10mM HEPES 143UmL benzylpenicillin potassium100mgmL streptomycin sulfate and 24mM NaHCO

3 pH

71) containing 10 FBS 1 PS and incubated at 37∘C under5 CO

2atmosphere [14]

24 Cell Proliferation Assay Theeffects of compounds on cellgrowths were according to the MTT assay procedures [15]The method is based on the ability of a mitochondrial dehy-drogenase from viable cells to cleave the tetrazolium ringsof the pale yellow MTT and form impermeable crystals ofa dark-blue formazan thus resulting in accumulation withinhealthy cells Briefly cells were seeded in 96-well plates at adensity of 8 times 103 cellswell The medium was then changedand cells were maintained in either solvent alone (controlcells) or in the presence of the indicated each sample in a finalvolume of 100 120583L in 10 FBS culture medium Each samplewas added to a microplate and incubated under the sameconditions as above for 24 h After 24 h of incubation themediumwas replaced with 100 120583L of freshmedium including05mgmL MTT The plate was cultured in a 37∘C incubatorfilled with 5 CO

2for 2 h Each precipitate in a specific

dish was dissolved in 100 120583L of DMSO to dissolve the purpleformazan crystals After the dishes were gently shaken for10min in the dark to ensuremaximal dissolution of formazancrystals the absorbance (119860) values of the supernatant were


0

30

60

90

120

Control 1 5 10 25 50 100

A20

58 ce

ll vi

abili

ty (

of c

ontr

ol )

BHPHTO (120583M)

lowastlowast

lowast

(a)

0

30

60

90

120

Control 1 5 10 25 50 100

A20

58 ce

ll vi

abili

ty (

of c

ontr

ol)

BDMC (120583M)

lowast

lowast

lowast

(b)







0

20

40

60

80

100

120

Control 1 5 10 25 50 100

Cel

l via

bilit

y (

of c

ontro

l)

BHPHTO (120583M)

lowast lowast

lowast

lowast

(a)

Control 1 5 10 25 50 100BDMC (120583M)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowast

lowast

lowast

(b)







0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BHPHTO (120583M)

lowastlowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BDMC (120583M)

lowastlowast

(b)





0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BHPHTO (120583M)

lowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BDMC (120583M)

lowast

lowastlowast

lowast

(b)








Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

30

60

90

120

Control 1 5 10 25 50 100

A20

58 ce

ll vi

abili

ty (

of c

ontr

ol )

BHPHTO (120583M)

lowastlowast

lowast

(a)

0

30

60

90

120

Control 1 5 10 25 50 100

A20

58 ce

ll vi

abili

ty (

of c

ontr

ol)

BDMC (120583M)

lowast

lowast

lowast

(b)







0

20

40

60

80

100

120

Control 1 5 10 25 50 100

Cel

l via

bilit

y (

of c

ontro

l)

BHPHTO (120583M)

lowast lowast

lowast

lowast

(a)

Control 1 5 10 25 50 100BDMC (120583M)

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowast

lowast

lowast

lowast

(b)







0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BHPHTO (120583M)

lowastlowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BDMC (120583M)

lowastlowast

(b)





0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BHPHTO (120583M)

lowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BDMC (120583M)

lowast

lowastlowast

lowast

(b)








Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BHPHTO (120583M)

lowastlowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Tyro

sinas

e act

ivity

( o

f con

trol)

BDMC (120583M)

lowastlowast

(b)





0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BHPHTO (120583M)

lowast

(a)

0

30

60

90

120

150

Control 1 5 10 25 50

Mela

nin

cont

ent (

o

f con

trol)

BDMC (120583M)

lowast

lowastlowast

lowast

(b)








Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Acknowledgment


References


























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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